Proteolytic N-terminal processing of Mfa proteins in the periodontal pathogen Porphyromonas gingivalis

The asaccharolytic bacterium Porphyromonas gingivalis regulates biofilm formation through Mfa1 fimbriae, composed of the major subunit Mfa1 and accessory proteins including the putative tip adhesin Mfa4. These components undergo maturation via N-terminal leader peptide cleavage by gingipains. However, the mechanisms governing fimbrial assembly remain unclear. This study examined the role of protease-dependent N-terminal processing in the maturation and incorporation of Mfa1 and Mfa4 during fimbrial biogenesis.

Missense mutations were introduced in the N-terminal regions of mfa1 and mfa4 by substituting RgpA/B- and Kgp-specific cleavage sites with alanine. Surface expression of Mfa1 in mutant cells was analyzed using ELISA. Mfa1 fimbriae were purified from parental and mutant strains via ion-exchange chromatography, and N-terminal sequences of Mfa1 and Mfa4 were determined. Antibodies targeting the Mfa4 leader peptide were used for localization studies.

Despite alanine substitutions at RgpA/B cleavage sites, Mfa1 processing persisted, indicating compensatory cleavage by Kgp or other enzymes such as dipeptidyl peptidases. Mature Mfa1 was transported to the cell surface and incorporated into fimbriae. Only the mature form of Mfa4 was detected in the fimbriae, whereas the leader peptide was enriched in the inner membrane.

These results suggest the existence of a compensatory proteolytic network in P. gingivalis and emphasize the biological importance of post-translational modifications in fimbrial assembly.